Hospital-acquired infections (HAIs) previously focused mainly on multidrug-resistant (MDR) bacteria, with less attention on viruses. The COVID-19 pandemic highlighted the importance of controlling viral infections. Human rhinoviruses (HRVs) are among the viruses responsible for HAIs. HRVs are non-enveloped viruses that infect the upper airways after airborne or direct transmission. Due to their lack of a membrane envelope, HRVs exhibit moderate resistance to commonly applied alcoholic disinfectants. Therefore, there is a significant need to develop alternative disinfection and hand sanitation strategies to control HRV infections in healthcare settings without posing a risk to human health. The antimicrobial activity and safety of rhamnolipids and rhamnolipids nano-micelles (RMN) against MDR-bacteria and several viruses, including SARS-CoV-2, were confirmed recently. Also, we previously demonstrated the superior antimicrobial activity of RMN over rhamnolipids. In the current study, molecular docking demonstrated the weak interactions of rhamnolipids with HRV-1A (minor group) compared to HRV-14 (major group), suggesting a superior antiviral activity of rhamnolipids towards major group rhinoviruses. To biologically validate these data, RMN was prepared and characterized, and then antiviral activity against HRV-16 (major group) and HRV-1B (minor group) infection of HeLa cells was assessed. RMN showed a complete inhibition of HRV-16 infection with recovery of 100% of HeLa cell viability. In contrast, only partial inhibition of HRV-1B infection with approximately 50% protection against infection was observed. Therefore, RMN might be recommended as a disinfectant and/or a hand sanitizer component to control the spread of RVs in hospital care settings or elsewhere to reduce the incidence of respiratory infections.